The automatic analysis of DNA is mainly based on the examination of the messenger RNA which controls the way in which the various parts of the genes are activated or deactivated to create certain types of cells.
If the gene is expressed in a single way, it can generate a normal muscular cell, while if it is expressed in another way it can generate a tumour.
By comparing the different expressions of genes, researchers aim at discovering the way to predict and prevent cancer.
Another possible application is the so-called pharmacogenomics, a discipline in which scientists attempt to correlate the smallest DNA variations of a person with reaction to various substances, such as drugs.
Numerous other possible applications are being implemented, developed and studied.
Over the past years, a technique based on the use of so-called DNA chips has been developed to allow automatic DNA analysis.
Essentially, DNA chips are small flat surfaces on which some rows, called probes, of one half of the double helix of DNA, are deposited according to a typical matrix configuration.
Since each half of the double helix of DNA is naturally bonded to its complementary other half in a process called hybridisation, the DNA chip can be used to identify the presence of particular genes in a biological specimen.
These chips are called microarrays in relation to their matrix structure, which may also be linear, and can be made employing different technologies, including semiconductor technology, on a variety of surfaces, including glass and plastic.
The use of DNA microarrays to delineate the expression of genes is the most important application of “biochips”. This method has completely replaced the previous methods which had the disadvantage of needing to be repeated either on each gene or on a restricted number of genes and were also difficult to automate.
For a general illustration of a possible application of these methods, useful reference can be made to the work by DeRisi J et. al., “Use of cDNA microarray to analyse gene expression patterns in human cancer”, NatGenet December 1996; 14 (4), 457–60.
Usually, DNA microarrays are used as interconnected memory chips in order to compare specimens of DNA from a patient against known, preserved specimens of DNA.
This is because DNA carries an electrical charge and this charge can be read on a chip, exactly in the way that occurs in a cell in a matrix of memory cells.
In many DNA chips, the coupling of arrays of DNA is signalled by means of fluorescent materials.
Notwithstanding, the procedure for analysing the chip, in particular to detect the levels of fluorescence, is rather costly.
Various methods have been developed to avoid these problems.
For example, according to a known solution, developed by the company Micro Sensors in collaboration with the company Motorola, DNA probe coupling is detected by means of bio-electronic methods.
This solution essentially consists in depositing a number from 10 to 50 DNA probes on a printed circuit.
An organic atom containing iron which can generate an electronic signal when the DNA rows are coupled, is used instead of fluorescence.
Parallel methods, allowing the simultaneous quantification of the level of expression of a very high number of genes by means of simultaneous querying, with a high sensitivity and fidelity of acknowledgement of the expression profile of a complete library of genes, have been developed over the past years.
With a certain degree of approximation, yet essentially close to reality, the methods based on the use of microarrays of genes can be ideally related to some main classes.
The method developed by Prof. Brown represents a first class of solutions. This method permits, by means of robot micro-machining, to chemically immobilize in 2 by 2 cm micro-grids fragments of cDNA (complementary DNA), or DNA reconstructed on the basis of RNA by reverse transcription. In this way, microarrays containing 10,000 individual cDNA elements are formed. The DNA fragment to be analyzed is marked with fluorescent groups so to obtain different types of sensors to immediately distinguish the fragments of DNA by means of the color of the corresponding fluorescent group with which they are treated. In this way, the microarray can be analyzed simultaneously during the hybridisation phase. The micro-grid is read by means of a confocal microscope at the end of the hybridisation phase providing a two-dimensional image in which colored pins, or spots, appear arranged in a grid. The intensity of the various colors and their combinations is directly correlated to the intensity of the light output by fluorescence by the respective probes and to the degree of affinity between the probes and the individual genes deposited on the grid.
Another technique is known as micro-spotting. In this technique, a robot arm is dipped in a DNA material in correspondence with an array of pins which is then impressed on a glass support.
Another method based on the use of microarrays was introduced by Affymatrix. This technique employs synthetic oligonucleotides, instead of natural fragments of DNA for constructing the micro-grid. These fragments are deposited on the grid by means of photolithography. In particular, masks for exposing some parts of a glass wafer on which certain chemical processes occur are used to make single row DNA sensors.
The use of photolithography, in combination with the chemical synthesis of oligonucleotides, results in a presence of approximately 100,000 genes in a single microarray which, according to current estimates, compose the complete library of mapped human characteristics.
The methods described provide as a final result an image which expresses the degree of genic expression in a fragment of DNA to be analysed by means of shades of different colors or combinations of colors.
The main advantage of the microarray method consists in the possibility of simultaneously analysing an extremely high number of genes.
This is because the information associated with the different cells present on the DNA chip can be processed in parallel with the consequent possibility of increasing the number of cells in the microarray to values in the order of 10,000–100,000 cells.
In this way, systems for the automated analysis of fragments of DNA can be provided which employ processing techniques of the images derived from microarray after hybridisation.
This notwithstanding, the systems of this type implemented to date are based on the analysis of very large images (a number of pixels which is one to two orders of magnitude greater than the number of cells which form the micro-grid). These images can be acquired in parallel, but are transferred and processed in a sequential fashion, as usually occurs in analysis techniques employing digital microprocessors, whereby processing speed is considerably penalised.
Consequently, the idea of using a DNA chip has not been fully exploited to date, due to the difficulty in achieving real time analysis of the respective fluorescent images. Moreover, since diagnostic protocols generally require a certain number of microarray-based experiments, the time required for analysing the resulting images (processing times in the range of 10 to 30 minutes) abnormally hinder the efficacy of such method.